The present invention relates to improved methods, reagents and kits for quantitating the expression of HLA-DR on the surface of human peripheral blood cells, particularly on the surface of peripheral blood monocytes. The invention further relates to methods for assessing immune competence and for directing and monitoring immunostimulatory therapies for sepsis based upon the levels of monocyte HLA-DR expression so measured.
Sepsis is one of the most common causes of death in developed countries. Incidence is increasing, and mortality remains high.
Early efforts to understand sepsis and to intervene in the progressive multiorgan failure of septic shock focused upon readily observable physical, physiologic, and anatomic symptoms. To this day, the acute management of fever, infection, coagulatory dysfunction, vascular collapse and end-organ failure remains the standard of care.
More recent efforts, however, have focused upon immunologic mediators thought to underlie these. systemic processes.
Animal models of sepsis have, for example, implicated a number of cytokines as mediators of the systemic inflammatory response seen early in the septic patient. In these models, acute parenteral challenge with endotoxin or with gram negative bacteria leads to production of tumor necrosis factor xcex1 (TNFxcex1), interleukin-1 (IL-1), and gamma interferon (IFNxcex3). Gamma interferon has been shown to act synergistically with TNFxcex1 in inducing shock in these animals.
Yet recent efforts to treat sepsis by immunomodulation have proven disappointing. Attempts to reduce or ablate the effects of proinflammatory cytokines, particularly TNFxcex1 and IL-1, have not only failed to improve outcome, but have in several cases increased mortality. Fisher et al., JAMA 271:1836-43 (1994); Fisher et al., N. Engl. J. Med. 334:1697-1702 (1996); Fisher et al., Crit. Care Med. 21:318-327 (1993); reviewed in Bone, JAMA 276:565 (1996). There thus exists a need for immunomodulatory therapies that improve clinical outcomes in sepsis.
Recently, several observations have motivated an alternative, seemingly contrarian, immunomodulatory approach.
Acute bacterial invasion is in fact an atypical clinical presentation for human sepsis. In most patients, sepsis is a late complication of trauma, burn, or major surgery. Infections in these patientsxe2x80x94coming late, as a secondary response to antecedent injury, rather than early, as the primary and actual cause of septic shockxe2x80x94bespeak a possible systemic immunosuppression or immune paralysis, rather than a state of hyperimmunity as predicted by the acute animal models.
In particular, studies have shown that HLA-DR expression by monocytes is severely depressed after trauma, and that such depressed levels correlate clinically with an increased susceptibility of trauma patients to infection. Polk et al., Ann. Surg. 204:282 (1986); Hershman et al., Clin. Exp. Immunol. 77:67-70 (1989).
Depression of monocytic HLA-DR expression has also been shown in a study of patients undergoing elective or emergent neurosurgery: patients who develop infectious complications in the postoperative period display a significantly lower level of monocytic HLA-DR expression than patients with an uncomplicated course; very low HLA-DR expression (fewer than 30 percent of peripheral blood monocytes positive for HLA-DR expression) predicts high risk for infection following surgery. Asadullah et al., Crit. Care. Med. 23:1976-1983 (1995).
Depression in HLA-DR expression has further been observed in the peripheral blood monocytes of septic patients with a wide variety of precipitating ailments. In these latter studies, surface immunophenotypic changes were further associated with decreased monocytic antigen-presenting function, reduced production of TNFxcex1, IL-1 and IL6, anergy, and alterations in lymphocyte activity. Volk et al., Behring Inst. Mitt. 88:209-215 (1991); Docke et al., in Reinhart et al. (eds.), Sepsis: Current Perspectives in Pathophysiology and Therapy, New York: Springer-Verlag (1994), pp 473-500.
Monocytes, like macrophages, B cells, and dendritic cells, are xe2x80x9cprofessionalxe2x80x9d antigen presenting cells (APCs). Although a number of cell types are capable of processing soluble antigens for subsequent display to T lymphocytes, the so-called xe2x80x9cnonprofessionalxe2x80x9d APCs lack the accessory molecules required to complete the process of T cell activation. xe2x80x9cProfessionalxe2x80x9d antigen-presenting cells, such as monocytes, not only process and present antigens in the context of MHC, but also possess the additional accessory molecules required to complete T cell activation, rendering them critical to the development of a full T cell-directed immune response. Reversal of monocytic deactivation in late-stage sepsis might, therefore, be expected to improve immune function, conferring clinical benefit.
Gamma interferon (IFNxcex3) is a major activator of monocytes. It upregulates the surface expression of costimulatory and HLA molecules, increasing monocyte antigen-presenting capacity, and primes for the LPS-induced production of proinflammatory cytokines. Young et al., J. Leukocyt. Biol. 58:373-381 (1995).
A single clinical trial of gamma interferon treatment of late-stage sepsis has been reported. Peripheral blood monocyte HLA-DR levels were monitored in patients meeting the inclusion criteria for severe sepsis. Gamma interferon was administered to those patients in whom, over two consecutive days, fewer than 30% of peripheral blood monocytes measured positive for HLA-DR expression. Treatment was continued until the percentage of monocytes with demonstrable HLA-DR expression remained over 50% for three days. Of the 10 patients, 8 showed an increase in monocyte HLA-DR expression within 1 day of treatment; the other 2 responded within 2 to 3 days. The recovery of monocytic HLA-DR expression was associated with restitution of monocytic function in vivo, as evidenced by a significant increase of TNFxcex1 and IL-6 plasma levels during treatment and a more favorable clinical outcome. Kox et al., Arch. Intern. Med. 157:389-393 (1997); Dxc3x6cke et al., Nature Med. 3:678-680 (1997).
Because administration of a proinflammatory cytokine would be contraindicated, however, in the early, hyperimmune phase of sepsis, there exists a need for a rapid, reliable method for measuring HLA-DR expression on peripheral blood monocytes. There further exists a need for a method that would report values for a given peripheral blood sample that are reliably and substantially independent of the individual testing laboratory.
Typically, as in the reported clinical study, monocyte HLA-DR expression is assessed flow cytometrically. Monocytes are distinguished from other peripheral blood cells by either surface immunophenotype, physical properties (side scatter and/or forward scatter), or some combination thereof; HLA-DR levels are assessed on the monocytes so distinguished by use of a fluorophore-conjugated anti-HLA-DR antibody.
Monocytes may, for example, be distinguished using an antibody specific for CD14. CD14, a receptor for lipopolysaccharide, is expressed predominantly on cells of the myelomonocytic lineage; in peripheral blood, CD14 is expressed principally by monocytes. But granulocytes in the blood also react with anti-CD14 antibodies, albeit weakly, and with present anti-CD14 conjugates cannot be completely discriminated from monocytes. Gating out CD14dim cells, as a means of removing granulocytes from the analysis, removes CD14dim monocytes as well, confounding the HLA-DR analysis. There thus exists a need for a fluorophore that, when conjugated to anti-CD14 antibody, would permit the immunocytochemical discrimination of monocytes from granulocytes.
HLA-DR may readily be labeled on the surface of peripheral blood cells, including monocytes, using a fluorophore-conjugated anti-HLA-DR antibody. But the surface expression of HLA-DR on the surface of monocytes is not a simple, static, and stable phenotype. MHC restriction imposes conflicting demands on the protein processing machinery of the APC: on the one hand, there is a requirement for proteolytically-processed peptide antigen; on the other, there is a requirement for intact MHC class II protein. These concurrent requirements are met by a finely choreographed, and as yet incompletely understood, coordinated movement of endocytosed antigen and newly-synthesized MHC class II molecules through various internal compartments of the cell. Cresswell, Annu. Rev. Immunol. 12:259-93 (1994). Rapid recycling of class II molecules from the surface, through compartments in part distinct from those traversed by newly-synthesized MHC, and then back to the surface, implicates yet other, likely intersecting, intracellular pathways. Reid et al., Nature 346:655-657 (1990); Roche et al., Proc. Natl. Acad. Sci. USA 90:8581-85 (1993); Watts, Annu. Rev. Immunol. 15:821-50 (1997).
The level of HLA-DR-specific fluorescence reported by peripheral blood monocytes depends upon the duration of incubation with anti-HLA-DR antibody, evidence of the dynamic nature of HLA-DR expression. This time dependence makes reliable absolute measurements of HLA-DR expression difficult. There thus exists a need for a method and reagents that would permit the stabilization of HLA-DR levels for flow cytometric assay.
The present invention solves these and other problems in the art by providing improved methods, reagents, and kits for quantitating the expression of HLA-DR on the surface of human peripheral blood cells, particularly on the surface of peripheral blood monocytes.
The invention is based, in part, upon the novel finding that inclusion of chloroquine during staining of peripheral blood with anti-HLA antibody stabilizes the HLA-DR-specific signal obtained from CD14+ monocytes. This is surprising: chloroquine has been reported to be unable to block the cellular recycling of MHC glycoproteins, and other agents that affect protein processing, such as Brefeldin A and monensin, have been shown ineffective in stabilizing HLA-DR expression.
The invention is also based in part upon the finding that conjugation of an anti-CD14 antibody to a fluorescence energy resonance transfer tandem fluorophore, PerCP/CY5.5, provides a high intensity, highly uniform and clustered signal from peripheral blood monocytes, permitting the ready discrimination of monocytes from granulocytes. Incorporating these improvements provides an assay well-suited for clinical use, permitting the rapid and reliable quantitative measurement of peripheral monocytic HLA-DR levels.
In a first aspect, therefore, the present invention provides a method of measuring HLA-DR expression on the surface of human blood cells, comprising contacting a sample containing human-blood cells with a lysosomotropic amine and an antibody specific for HLA-DR, and then detecting the binding of the anti-HLA-DR antibody to the cells. In preferred embodiments, the lysosomotropic amine is chloroquine. In preferred embodiments, the anti-HLA-DR antibody is conjugated to a fluorophore, preferably at a defined molar ratio; particularly preferred is conjugation to PE at a defined molar ratio, and especially preferred is conjugation to PE at a molar ratio of 1:1.
The invention also provides for the measurement of HLA-DR on the surface of peripheral blood monocytes. In preferred embodiments, a monocyte-distinguishing antibody, typically anti-CD14, is included in the staining step. In particularly preferred embodiments, the anti-CD14 antibody is conjugated to the PerCP moiety of a PerCP/CY5.5 tandem fluorophore molecule, permitting single-laser, multicolor flow cytometric analysis.
In preferred embodiments, a whole blood sample is employed, although the methods and reagents may equally be used with appropriate blood fractions, such as fractions enriched in peripheral blood mononuclear cells. In the whole blood embodiments, a further step of lysing the erythrocytes in the blood sample preferably intervenes between the staining step and the detection step. The method may include yet a further step of removing lysis debris after the lysis step but before detection.
Results from the improved HLA-DR assay of the present invention correlate well with results from prior, but problematic, assays. This high degree of correlation permits the direct translation of the established clinical criteria to measurements reported by the present assay.
Thus, in another aspect, the present invention provides a method of assessing the immune status of a human patient, comprising the steps of contacting a sample containing the patient""s blood cells with a lysosomotropic amine and an antibody specific for HLA-DR; detecting the binding of the anti-HLA-DR antibody to the monocytes in the sample; and then comparing the level of binding so detected with that so detected from human controls. In preferred embodiments of this method, the lysosomotropic amine is chloroquine, the anti-HLA-DR antibody is conjugated to PE, and a monocyte-distinguishing antibody, such as anti-CD14, preferably anti-CD14-PerCP/CY5.5, is included in the staining step.
The invention also provides, in a related aspect, a method for determining the suitability of immunostimulatory therapy in a patient with sepsis, comprising contacting a sample containing the patient""s blood cells with a lysosomotropic amine and an antibody specific for HLA-DR; detecting the binding of the anti-HLA-DR antibody to the monocytes in the sample; and then comparing the level of binding so detected with that detected from normal controls. Patients with levels of binding less than that of controls are determined to be suitable for immunostimulatory treatment. Applying present clinical criteria, sepsis patients averaging fewer than 5000 anti-HLA-DR antibodies per monocyte are determined to be suitable for immunostimulatory treatment, with patients averaging fewer than 3000 anti-HLA-DR antibodies per monocyte determined to be particularly suitable, and with patients averaging fewer than 3000 anti-HLA-DR antibodies per monocyte for two consecutive days determined to be especially suitable for immunostimulatory treatment.
In especially preferred embodiments of the method for determining the suitability of treatment, a peripheral blood sample is in the first step contacted with an anti-HLA-DR PE antibody, an anti-CD14-PerCP/CY5.5 antibody, and chloroquine.
In another aspect, the present invention provides compositions for performing the subject methods. The compositions for flow cytometric measurement of HLA-DR on human peripheral blood cells comprise a fluorophore-conjugated anti-HLA-DR antibody and a lysosomotropic amine, preferably chloroquine. The anti-HLA-DR antibody is preferably conjugated to PE, most preferably at a defined molar ratio, and in especially preferred embodiments, at a molar ratio of 1:1.
For flow cytometric measurement of HLA-DR on peripheral blood monocytes, the compositions of the present invention further comprise a monocyte-distinguishing antibody, preferably an anti-CD14 antibody, more preferably an anti-CD14 antibody conjugated to a fluorescence energy transfer tandem fluorophore, most preferably conjugated to the PerCP moiety of PerCP/CY5.5.
The present methods are particularly useful in clinical settings and will thus likely be performed, inter alia, by clinical laboratories. However, any one clinical laboratory may have only sporadic need to perform such methods. The invention thus provides, in another aspect, kits that permit the assay readily to be performed on an as-needed basis.
In preferred embodiments, a kit of the present invention comprises one or more staining compositions and an erythrocyte-lysing composition. At least one staining composition comprises an anti-HLA-DR antibody and a lysosomotropic amine, preferably chloroquine. In preferred embodiments, the anti-HLA-DR antibody is conjugated to PE, preferably at a defined molar ratio, most preferably at a ratio of 1:1. In embodiments useful for quantitating HLA-DR on the surface of peripheral blood monocytes, at least one staining composition of the kit comprises a monocyte-distinguishing antibody, preferably anti-CD14. In preferred embodiments, the anti-CD14 antibody is conjugated to a fluorescence resonance transfer tandem fluorophore, most preferably PerCP/CY5.5. In particularly preferred embodiments, the kit contains a staining composition that comprises an anti-HLA-DR-PE antibody, an anti-CD14-PerCP/CY5.5 antibody, and chloroquine.
In other embodiments, the kit comprises one or more staining compositions, an erythrocyte lysing composition, and further comprises pelletized beads conjugated with defined levels of PE. In these embodiments, the anti-HLA-DR antibody is conjugated to PE and the pelletized beads permit the calibration of the flow cytometer to allow quantitation, from the measured PE fluorescence, of the amount of HLA-DR antibody bound per cell.
In another aspect, the invention provides a novel monocyte-specific immunoconjugate, comprising an anti-CD14 antibody conjugated to the PerCP moiety of a PerCP/CY5.5 tandem dye molecule. The specificity of the CY5.5 moiety for CD64, coupled with the specificity of the antibody for CD14, provides a high intensity monocyte-specific signal for single laser, multicolor fluorescence activated cell sorting applications.